August 7, 2020

Protein Biogenesis Demands of the Early Secretory Pathway during Heterologous Expression

Previous findings

Overall design of new experiment

Preliminary data

Weissman/Ingolia method

McGlincy and Ingolia (2017)

  • Used in Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

  • Successful in 9/20 attempts (all 8 samples in HSA expression study had rRNA contamination)

1: Prepare a cell lysate

2: Nuclease footprinting and ribosome recovery

McGlincy and Ingolia (2017)

2: Nuclease footprinting and ribosome recovery

3: Footprint fragment purification

McGlincy and Ingolia (2017)

3: Footprint fragment purification

4: Dephosphorylation and linker ligation*

McGlincy and Ingolia (2017)

4: Dephosphorylation and linker ligation*

5: rRNA reduction*

McGlincy and Ingolia (2017)

5: rRNA reduction*

6: Reverse transcription

McGlincy and Ingolia (2017)

6: Reverse transcription

7: Circularization

McGlincy and Ingolia (2017)

7: Circularization

8: Library construction PCR

McGlincy and Ingolia (2017)

8: Library construction PCR

amp

Possible adaptions

4: Dephosporylation and poly adenylation

This experiment

  • NEB’s E. coli poly(A) polymerase is meant to be incubated at 37˚C while other protocols use a similar enzyme at 16˚C (Hornstein et al. (2016)), can we get away with lower temperatures to better for slower rate of adenylation?

  • At lower temperatures, what time frame are we looking at to add 15 A’s (approximate length of reverse transcription primer)?

What went wrong?

  1. Control concentration was diluted relative to range of sample concentrations

  2. Gel ran too long (dye front)

  3. Ethanol precipitation failed (was not mixed properly)

This experiment

  • Incubated 18nt control at 16˚C for 5, 6, and 7 minutes (only had enough enzyme for three runs) based on previous gel

  • Duplicate ULR was to see if formamide buffer degrades ladder

  • Duplicate markers were to test old and new marker to see if there are more than 2 bands

Next experiments

  • Make sure 34 nt pure oligo also has higher weight band (I believe these are forming homodimers)

  • Incubate at 16˚C for 8, 10, 12, 14, minutes
    • For best time, do this with 18 nt and 34 nt markers to make sure there is no fragment length bias
  • Other variables that affect poly adenylation:
    • ATP concentration
    • Poly A enzyme concentration
    • RNA input concentration

5: Reverse transcription with TSO

New England Biolabs (n.d.)

6: rRNA reduction with double stranded nuclease

Chung et al. (2015)

References

Chung, Betty Y, Thomas J Hardcastle, Joshua D Jones, Nerea Irigoyen, Andrew E Firth, David C Baulcombe, and Ian Brierley. 2015. “The Use of Duplex-Specific Nuclease in Ribosome Profiling and a User-Friendly Software Package for Ribo-Seq Data Analysis.” RNA 21 (10): 1731–45.

Hornstein, Nicholas, Daniela Torres, Sohani Das Sharma, Guomei Tang, Peter Canoll, and Peter A Sims. 2016. “Ligation-Free Ribosome Profiling of Cell Type-Specific Translation in the Brain.” Genome Biol. 17 (1): 149.

McGlincy, Nicholas J, and Nicholas T Ingolia. 2017. “Transcriptome-Wide Measurement of Translation by Ribosome Profiling.” Methods, June.